Whole Genome High-Throughput Screen Identified microRNAs Enhancing rAAV Production

By B. Finkbeiner, M. Burkhart, S. Reichl, R. Derler, A. Schulze, F. Sonntag, K. Otte, M. Hoerer

Recombinant adeno-associated viruses (rAAV) have emerged as the leading vector for gene therapy in recent years. However, manufacturing bottlenecks present a significant challenge in meeting the growing clinical and commercial demand for high-quality rAAV. While efforts have focused on scaling up vector production and expanding manufacturing capacity, strategies to enhance cell-specific and volumetric rAAV productivity remain underutilized, despite their potential to improve both yield and quality.

MicroRNAs (miRNAs), short non-coding RNAs that regulate gene expression across various cellular processes, have already been successfully leveraged to enhance the production of conventional biologics. As a result, miRNAs are seen as a promising tool to address critical limitations in rAAV vector production, potentially driving efficiency to the necessary levels.

Using the Ascend HEK293 cell line and a split two-plasmid system, we have developed a robust, modular suspension platform for rAAV production, optimized to maximize yield while maintaining consistent quality from bench scale to 200 L. To systematically identify host cell factors that significantly influence rAAV productivity in our HEK293 cells, we conducted a high-throughput screen utilizing a genome-wide library of human microRNA mimics.